American Journal of Medical Genetics (Neuropsychiatric Genetics) 67:488490 (1996)
Brief Research Communication
Lack of Association Between
TaqI
A1 Allele
of Dopamine D2 Receptor Gene and Alcohol-Use
Disorders in Atayal Natives
of
Taiwan
Chia-Hsiang Chen, Shih-Hsiang Chien, and Hai-Gwo Hwu
Division of Psychiatry, Cheng Hsin Rehabilitation and Medical Center, Taipei (C.-H.C.); Division of Neuropsychiatry, School of Medicine, National Yang Ming university, Taipei (C.-H.C.); Department of Psychiatry, National Taiwan University, Taipei, Taiwan (S.-H.C., H.-G.H.)
Association studies between the A1 allele of the dopamine D2 receptor (DRDS) gene TuqI A polymorphism and alcoholism remain controversial. A recent study from Japan demonstrated that the A1 allele is associ- ated with severe alcoholism in the Japanese population. We were interested in knowing if this association also exists in the Atayals of Taiwan, who were found to have a higher prevalence of alcohol-use disorders than the Han Chinese in Taiwan. Genotype and allele frequencies were determined in alcohol- abusing, alcohol-dependent, and nonalco- holic control Atayal natives in Taiwan. A1 allele frequencies in alcohol-dependent, alcohol-abusing, and normal control Atayals were 0.39,0.42, and 0.39, respectively. No dif- ference in A1 allele frequency was found among these three groups. Our data do not support the hypothesis that the A1 allele of the TuqI A polymorphism of the DRD2 gene increases susceptibility to alcohol-use disor- ders in the Atayals of Taiwan.
0 1996 Wiley-Liss, Inc.
KEY WORDS: restriction fragment length polymorphism, susceptible gene, association
INTRODUCTION
Alcoholism is a complex, multifactorial disease, with both environmental and biological origins. Family,
Received for publication November 20, 1995; revision received March 18, 1996.
Address reprint requests to Dr. Hai-Gwo Hwu, Department of Psychiatry, National Taiwan University Hospital, 7 Chung-Shan S. Rd., Taipei 100, Taiwan.
0 1996 Wiley-Liss, Inc.
twin, and adoption studies have elucidated the impor- tance of the genetic component of alcoholism [Merikan- gas, 1990; Pickens e t al., 1991; Kendler e t al., 19921. However, no gene has yet been identified which in- creases susceptibility to alcoholism. Blum et al. [ 1990, 19911 first reported a higher frequency of the A1 allele of TuqI A polymorphism of the DRDB gene in alcoholics compared to nonalcoholic controls. This finding implied that a molecular variant of the DRD2 gene may in- crease susceptibility to alcoholism. Since neurobiologi- cal studies have provided evidence that alcohol works on the central dopaminergic system, which is involved in drug-mediated reinforcement behavior, and which may be related to the pathogenesis of alcoholism, many researchers have tried to replicate the initial observa- tions of Blum et al. [1990]. Some groups were able to find the association [Comings e t al., 1991; Parsian e t al., 1991; Amadeo et al., 1993; Neiswanger et al., 1995al. However, many groups were unable to obtain the same result [Gelernter et al., 1993; Noble, 19931. Therefore, the finding of allelic association between DRDB TaqI A and alcoholism remains controversial.
Atayal natives of Taiwan are aboriginal people of Malayo-Polynesian heritage, who colonized Taiwan several centuries before the arrival of Han Chinese im- migrants from mainland China. Previous epidemiolog- ical studies have revealed a higher prevalence of alco- hol abuse (11.6%) and alcohol dependence (11.4%) among Atayals than Han Chinese in Taipei (3.4% for alcohol abuse, and 1.5% for alcohol dependence) [Hwu et al., 19901, which is a serious social and family issue among the Atayals of Taiwan. Recently, a n association study between the A1 allele of DRD2 TaqI A polymor- phism and alcoholism carried out in the Japanese pop- ulation demonstrated the existence of a n association between the A1 allele and severe alcoholism [Arinami et al., 19931. The severity of alcoholism increased in the order of the genotypes A2lA2, AllA2, and AlIA1 in Japanese alcoholics. We were interested in under- standing if the association and correlation between the A1 allele and alcoholism also exists in Atayal natives of
DRD2 and Alcoholism in Atayals of Taiwan 489
TaqI A polymorphism and alcohol-use disorders, were
evaluated by chi-square test.
RESULTS
The genotype and allele frequencies of the TaqI A polymorphism of the DRD2 gene of the three groups are listed in Table I. The distribution of DRDB genotypes of the three groups, namely alcohol-dependent
(x2
= 2.36, df = 1, P = 0.121, alcohol-abusing (x2 = 0.31, df = 1,P = 0.58), and nonalcoholic control (x2 = 0.67, df = 1,
P = 0.41), did not depart from the Hardy-Weinber equilibrium. There was no difference in genotype
(x
= 0.08, df = 2, P = 0.96) and allele frequencies
( x 2
= 0.00, df = 1, P = 1.00) between the alcohol-dependent group and normal controls. Also, no difference in geno- type (x2 = 0.40, df = 2, P = 0.82) and allele frequencies(x
= 0.13, df = 1, P = 0.71) was detected between the alcohol-abusing group and normal controls.8
DISCUSSION
In this study, we found no evidence to support an as- sociation between the A1 allele of TaqI A polymorphism of the DRDB gene and alcoholism in a genetically iso- lated aboriginal population, the Atayals, of Taiwan. Our results are in line with several other research groups, who also did not find an association between the A1 al- lele and alcoholism [reviewed in Gelernter et al., 19931. As discussed by many researchers, association studies are liable to errors of sampling bias and different allele distributions in different ethnic groups. In the present study, our subjects were homogeneous in ethnic back- ground, and lived in an isolated geographic area, avoid- ing possible population stratification. Moreover, our sample size was large enough to provide enough power to detect an association, if present.
The A1 allele frequency of normal Atayals of Taiwan is about 0.39, which is comparable t o Han Chinese normal controls (0.37) (our unpublished data) and Japanese normal controls (0.42) [Arinami et al., 19931. Orientals seem to have a significantly higher A1 allele frequency than Caucasians. However, unlike the study which reported an association between the A1 allele of the DRDB gene and severe alcoholism in the Japanese population [Arinami et al., 19931, no association was found in our study. The reason for the discrepancy may Taiwan. To address this issue, genotype and allele fre-
quencies of the TaqI A polymorphism of Atayals with alcohol-use disorders and of nonalcoholic Atayal con- trols were determined in this study.
MATERIALS
AND
METHODS SubjectsSubjects were Atayal people who participated in our serial Taiwan Aborigine Alcoholism Studies. These in- dividuals were enrolled with informed consent, and were interviewed by well-trained interviewers using the section on alcoholism of the modified Chinese Diag- nostic Interview Schedule [Hwu et al., 1990; Lin et al., 19951, which was shown t o have satisfactory interrater reliability. The diagnosis of alcohol-use disorders, i.e., alcohol dependence and alcohol abuse, was based on clinical assessment according t o the criteria of DSM- 111-R criteria. Nonalcoholic controls were also Atayals living in the same community, and they were evaluated by the interviewers to exclude the diagnosis of alcohol- use disorder, but not other neuropsychiatric disorders. One hundred and ninety-nine unrelated Atayals were recruited in this study, including 85 diagnosed as alcohol-abusing, 73 as alcohol-dependent, and 41 as nonalcoholic controls. The alcohol-abusing group con- sisted of 44 males and 41 females with a mean age of 40 years. The alcohol-dependent group consisted of 51 males and 22 females, with a mean age of 40.5 years. The non-alcoholic control group consisted of 15 males and 26 females, with an average age of 41 years.
Genotype Determination
Blood samples were collected with EDTA anticoagu- lant, and genomic DNA was prepared using the stan- dard method. Restriction fragment length polymorphism (RFLP) analysis of TaqI A polymorphism of the DRDB gene was carried out by using a PCR-based restriction analysis according to the method described by Grandy et al. [19931, with modifications. In brief, amplification reactions were carried out in a volume of 50 pl, contain- ing 100 ng genomic DNA as template, 200 pM dNTP, 1 pM of each sense and antisense primer, 1 X DynaZyme I1 reaction buffer (Finnzymes, Oy, Finland), 1.5 mM MgC12, and 1 unit of DynaZyme I1 Taq polymerase (Finnzymes,
Oy, Finland). The sequence of sense and antisense primers were 5'-CCGTCGACGGCTGGCCAAGTTGTCTA- 3', and
5'-CCGTCGACCCTTCCTGAGTGTCATCA-3',
respectively. After initial denaturation a t 94°C for 5 min, 30 cycles of PCR reaction were performed under conditions of denaturation at 94"C, 1 min, annealing at 56"C, 1 min, and extension a t 72"C, 1 min. Ten pl of PCR products were digested with 5 units of TaqI re- striction enzyme (Boehringer Mannheim, Germany) in buffer B in a total volume of 20 p1 at 65°C for a t least 6 hr. Digested PCR products were separated by electro- phoresis in a 2% Metaphor (FMC BioProducts, Rock- land) agarose gel, and visualized with ethidium bro- mide staining under ultraviolet light. The A1 allele shows a band of 310 bp, whereas the A2 allele shows bands of 180 bp and 130 bp.
Statistical analysis of the fitness of the Hardy- Weinberg equilibrium, and the allelic association of
TABLE I. Genotype and Allele Frequencies of TuqI A Polymorphism of the DRDB Gene
Among Atayal Natives of Taiwan* Allele
Genotypes frequency
AlIA1 A1lA2 A2JA2 N A1 A2
11% 56% 33% 39% 61%
16% 52% 32% 42% 58%
39% 61%
12% 54% 34%
* AD, alcohol dependence; AA, alcohol abuse; C, nonalcoholic control; N, number of individuals.
AD 8 41 24 73 57 87
AA 14 44 27 85 72 98
490 Chen et al.
lie in the differing severities of alcohol dependence of in our study and in the Japanese study of Arinami et al. [1993]. As Noble et al. [1994] reported that severity of alcohol dependence in alcoholics and of substance- abuse behaviors in controls are important variables in DRD2 allelic associations, our alcoholic subjects were recruited from the community, instead of from hospital, and were not evaluated for severity of alcohol depen- dence. Therefore, our alcoholic subjects may comprise patients with differing severities of alcohol depen- dence, who would be different from patients with se- vere medical complications recruited from hospitals. In addition, as appropriate controls are important in population-based association studies [Neiswanger et al., 1995b1, nonalcoholic controls with other neuro- psychiatric disorders were not excluded from our con- trol group in the present study. Thus, the real asso- ciation may be obscured. Even so, our results are consistent with the finding that no association exists between the A1 allele and alcoholism in Han Chinese alcoholics in Taiwan [Lu et al., 19931.
In summary, we did not find an association between the A1 allele of TuqI A polymorphism of the DRDB gene and alcohol-use disorders in Atayal natives of Taiwan. However, the association with severe alcohol depen- dence cannot be ruled out in the present study.
ACKNOWLEDGMENTS
This study was supported by grant NSC-84-2332-B- 002-250 from the National Science Council of Taiwan, R.O.C.
REFERENCES
Amadeo S, Fourcade ML, Abbar M, Leroux MG, Castelnau D, Venisse J L , Mallet J (1993): Association between D2 receptor gene poly- morphism and alcoholism. Psychiatr Genet 3:130.
Arinami T, Itokawa M, Komiyama T, Mitsushio H, Mori H, Mifune H, Hamaguchi H, Toru M (1993): Association between severity of al- coholism and the A1 allele of the dopamine D2 receptor gene Taql A RFLP in Japanese. Biol Psychiatry 33:10&114.
Blum K, Noble EP, Sheridan PJ, Montgomery A, Ritchie T, Jagadeeswaren P, Nogami H, Briggs AH, Cohen J B (1990): Allelic association of human dopamine D2 receptor gene in alcoholism. JAMA 263: 2055-2060.
Blum K, Noble EP, Sheridan P J , Finley 0, Montgomery A, Ritchie T, Ozkaragoz T, Fitch RJ, Sadlack F, Sheffield D, Dahlmann T, Halbardier S, Nogami H (1991): Association of the A1 allele of the D2 dopamine receptor gene with severe alcoholism. Alcohol 8: 4 0 9 4 1 6 .
Comings DE, Comings BG, Muhleman D, Dietz G, Shahbahrami B, Tast D, Knell E, Kocsis P, Baumgarten R, Kovacs BW, Levy DL, Smith M, Borison RL, Evans DD, Klein DN, MacMurray J , Tosk JM, Sverd J , Gysin R, Flanagan SD (1991): The dopamine D2 re- ceptor locus as a modifying gene in neuropsychiatric disorders. Gelernter J, Goldman D, Risch N (1993): The A1 allele at the D2 dopamine receptor gene and alcoholism, a reappraisal. JAMA 269: Grandy DK, Zhang Y, Civelli 0 (1993): PCR detection of the TaqA
RFLP at the DRDB locus. Hum Mol Genet 2:2197.
Hwu HG, Yeh YL, Wang JD, Yeh EK (1990): Alcoholism among Tai- wan aborigines defined by the Chinese Diagnostic Interview Schedule: A comparison with alcoholism among Chinese. Acta Psy- chiatr Scand 82:374-380.
Kendler KS, Heath AC, Neale MC, Kessler RC, Eaves L J (1992): A population based twin study of alcoholism in women. JAMA 268:1877-1882.
Lin L-S, Hwu H-G, Soong W-T, Yeh L-L, Chang C, Chang S-H, Lin S-T, Wang MT, Chen C-C (1995): Establishment of a n interview sched- ule for psychopathology of alcoholism. Chin Psychiatry 9:111-121. Lu R-B, Chang F-M, KO H-C, Castiglione CM, Schoolfield G, Kidd JR,
Kidd KK (1993): No association between alcoholism and the A1 a t DRDB gene in Han Chinese. Psychiatr Genet 3:172.
JAMA 26611793-1800.
1673-1677.
Merikangas KFt (1990): The genetic epidemiology of alcoholism. Psycho1 Med 2O:ll-22.
Neiswanger K, Hill SY, Kaplan BB (1995a): Association and linkage studies of the TuqI A1 allele at the dopamine D2 receptor gene in
a sample of female and male alcoholics. Am J Med Genet 60: Neiswanger K, Kaplan BB, Hill SY (199513): What can the DRD2I alcoholism story teach us about association studies in psychiatric genetics? Am J Med Genet 60:272-275.
Noble E P (1993): The D2 dopamine receptor gene: A review of associ- ation studies in alcoholism. Behav Genet 23:119-129.
Noble EP, Syndulko K, Fitch RJ, Ritchie T, Bohlman MC, Guth P, Sheridan P J , Montgomery A, Heinzmann C, Sparkes RS, Blum K (1994): D2 dopamine receptor TaqI A alleles in medically ill alco- holic and nonalcoholic patients. Alcohol Alcohol 29:729-744. Parsian A, Todd RD, Devor E J , OMalley KL, Suarez BK, Reich T,
Cloninger CR (1991): Alcoholism and alleles of the human D2 dopamine receptor locus: Studies of association and linkage. Arch Gen Psychiatry 48:655463.
Pickens RW, Svikis DS, McGue M, Lykken DT, Heston LL, Clayton PJ (1991): Heterogeneity in the inheritance of alcoholism. Arch Gen Psychiatry 43:19-28.
